L-NAME in the cardiovascular system – nitric oxide synthase activator?

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Review

L-NAME in the cardiovascular system – nitric oxide synthase activator?

Jana Kopincová

^1,2

, Angelika Púzserová

²

, Iveta Bernátová

²

1Department of Physiology, Jessenius Faculty of Medicine, Comenius University, Malá Hora 4A, 036 01 Martin, Slovak Republic

2Institute of Normal and Pathological Physiology, Centre of Excellence for Examination of Regulatory Role of Nitric Oxide in Civilization Diseases, Slovak Academy of Sciences, Sienkiewiczova 1, 813 71 Bratislava, Slovak Republic

Correspondence: Jana Kopincová, e-mail: jana.kopincova@jfmed.uniba.sk

Abstract:

L-arginine analogues are widely used inhibitors of nitric oxide synthase (NOS) activity both in vitro and in vivo, with N^w-nitro- L-arginine methyl ester (L-NAME) being at the head. On the one hand, acute and chronic L-NAME treatment leads to changes in blood pressure and vascular reactivity due to decreased nitric oxide (NO) bioavailability. However, lower doses of L-NAME may also activate NO production via feedback regulatory mechanisms if administered for longer time. Such L-NAME-induced activation has been observed in both NOS expression and activity and revealed considerable differences in regulatory mechanisms of NO production between particular tissues depending on the amount of L-NAME. Moreover, feedback activation of NO production by L-NAME seems to be regulated diversely under conditions of hypertension. This review summarizes the mechanisms of NOS regulation in order to better understand the apparent discrepancies found in the current literature.

Key words:

L-arginine analogues, N^w-nitro-L-arginine methyl ester, nitric oxide synthase, feedback regulation, nuclear factor kB

Abbreviations: BHR – borderline hypertensive rats, EDRF – endothelium-derived relaxing factor, eNOS – endothelial nitric oxide synthase, iNOS – inducible nitric oxide synthase, L-NA – N^w-nitro-L-arginine, L-NAME – N^w-nitro-L-arginine methyl ester, L-NMMA – N^w-monomethyl-L-arginine, NF-kB – nu- clear factor kB, NO – nitric oxide, NOS – nitric oxide syn- thase, ROS – reactive oxygen species, SHR – spontaneously hypertensive rats, WKY – Wistar-Kyoto rats

Introduction

The great and well-known nitric oxide (NO) story be- gan in 1987, when two experimental groups [31, 50]

finally identified chemical basis of endothelium- derived relaxing factor (EDRF) discovered formerly by Furchgott and Zawadzki in 1980 [21]. After 6-year long hard work of identifying EDRF, one year sufficed to reveal the intracellular source of NO, L-arginine [52]

and at the same time, L-arginine analogue, N

^w

-mono- methyl-L-arginine (L-NMMA), which had been shown to inhibit NO-synthesizing enzyme [51].

Discovery of three types of NO synthase (NOS) in early 90’s has brought interest to developing various inhibitors of NO synthesis. Soon, L-arginine ana- logues became widely used NOS inhibitors, provid- ing useful tool for achieving NO-deficient condi- tions.

Pharmacological Reports 2012, 64, 511520 ISSN 1734-1140

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Analogues of L-arginine serve as NOS inhibitors by virtue of their substitution at one or both of the termi- nal guanidino (

^G

or

^w

) nitrogen [68]. Generally, L-iso- mers solely are considered to be active inhibitors, al- though there is also evidence that N

^w

-nitro-D-arginine might inhibit endothelial relaxation of the aortal rings and N

^w

-nitro-D-arginine methyl ester inhibited NOS activity both in the rat heart and aorta [5, 83].

The mechanism of NOS inhibition by substrate analogues consists in competitive bonding to enzyme, but the molecular background of inhibitory action varies for particular analogues. In most cases, ana- logue-mediated inhibition of NO synthesis is in vivo reversible after L-arginine replenishment. However, there are some substances, such as L-NMMA, N

^w

- iminoethyl-L-ornithine or N

^w

-allyl-L-arginine that can be utilized by NOS forming intermediates tightly bound to enzyme molecule and thereby blocking the synthesis irreversibly – that is why they deserved the name “suicide inhibitors” [49, 68].

Reversible inhibitor and one of the most frequently used L-arginine substituents is N

^w

-nitro-L-arginine (L-NA), or its esterified form N

^w

-nitro-L-arginine methyl ester (L-NAME), which are considered to be non-selective inhibitors [43, 74]. Esterification of the carboxyl group of L-NA increases water solubility, which simplifies the experimental use of this ana- logue. On the other hand, specific esterase is needed to fully exhibit inhibitory action in the tissue and that may constrain the effect of L-NAME for particular tissues [27]. Despite 30–100-times lower inhibitory efficiency compared to L-NA, L-NAME is widely used in both acute and long-term in vitro and in vivo experiments, when the effects of NO production re- striction are investigated.

L-NAME administration

In endothelium, NO production is continual, keeping balance between vasoconstriction and vasodilatation.

NO acts as an antagonist of various constrictoric fac- tors with sympathetic nervous system at the head [2].

Therefore, changes in arterial blood pressure or total peripheral resistance after acute L-NAME administra- tion into vascular bed can be very quickly noticed and

tion of both L-NAME and L-NMMA led to dose- dependent constriction of the isolated rings of the rat aorta and femoral artery in vitro [64].

The theory of “endothelial dysfunction”, and con- sequential insufficient NO production in human es- sential hypertension, led to creation of an animal model of human hypertension due to NO deficiency, achieved by long-term L-NAME treatment of experi- mental animals. Long-term administration of NOS in- hibitor in relatively high doses (10, 20, 25, 40, 50, 65, 80, 100 mg per kg of body mass per day) induced so- called “NO-deficient hypertension” in normotensive rats and this model became widely used tool for in- vestigation of the NO participation in cardiovascular disorders [3, 11, 13, 18, 19, 35, 40, 48, 66, 86]. Never- theless, the way of drug administration, the dose as well as the treatment period varies from case to case and sometimes ends in arguable findings, as the ef- fects of L-NAME treatment on blood pressure may be inconsistent even in one laboratory using the same rat strain (compare [40, 48] and [77]).

Another controversial matter of long-term L-NAME treatment is L-NAME-induced left ventricular hyper- trophy. In some cases left ventricular hypertrophy was present after 3-week-long treatment with the L-NAME in the dose of 10 mg/kg/day, but, on the other hand, hy- pertrophy was absent even after 8 weeks of 100 mg/kg/

day L-NAME administration (Tab. 1).

Regarding vascular function, higher doses of L-NAME administered for 3–6 weeks reduced relaxant response of the aorta, femoral artery and small mesen- teric arteries to carbachol or acetylcholine in vitro [10, 19, 39, 57]. In addition, the doses of 10 to 100 mg of L-NAME per kg/day led to dramatic decrease of cGMP content in the aorta of normotensive rats [4, 13, 26].

One of very specific cases is an administration of

L-NAME to rat strain with spontaneous or borderline

hypertension. These spontaneously hypertensive rats

(SHR) develop blood pressure up to 180–200 mmHg

in the 4

^th

– 6

^th

week of life on genetic basis without

any pharmacological or physiological intervention,

regardless of sodium diet [69]. Borderline hyperten-

sive rats (BHR) are F1 offspring of one normotensive

and one spontaneously hypertensive parent. Such

genetic combination becomes manifested by blood

pressure elevation to levels about 130–150 mmHg [8,

67]. Previously, SHR were thought to have impaired

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NO production because of endothelial dysfunction observed in this strain [62, 85]. Later, many experi- ments showed elevated basal NO production in SHR [25, 63]. However, the organism dependence on NO in SHR is much higher than in normotensive strains supposedly due to elevated oxidative stress resulting in elevated NO turnover. Thus, in SHR L-NAME ad- ministration of 25 mg/kg/day for just 15 days had fa- tal consequences [1]. Moreover, 4-week L-NAME ad- ministration of 8–12 mg/kg/day led in SHR to blood pressure elevation up to 30–40 mmHg, increase in to- tal peripheral resistance, myocardial hypertrophy and diminished relaxation of the mesenteric vascular bed or the aorta to carbachol or isoprenaline, together with significant morbidity and mortality [6, 7, 47, 73, 78].

Within 4 weeks of ~8 mg/kg/day of L-NAME, only 42% of SHR survived and prolongation of treatment up to 2 months resulted in 100% morbidity/mortality in SHR [47]. Furthermore, in our experiments, ad- ministration of L-NAME in the low dose of 1.5 mg/

kg/day to SHR caused the death of animals within 2–4 weeks and marked blood pressure elevation was

observed even after the dose of 0.1 mg/kg/day in 2 weeks, while similar doses had no effect on blood pressure in normotensive rats [36].

Similar discrepancy was found in normotensive rats and BHR after 4 weeks of approximately 17 mg/kg/day L-NAME administration (drinking of L-NAME solu- tion with concentration 150 mg/l). The dose, which was tolerated by normotensive rats without any conse- quences after the withdrawal of treatment, led to sig- nificant blood pressure elevation in BHR even after the termination of drug administration, in some cases asso- ciated with lethargy, suppressed appetite and loss of motor function in the pelvic appendages followed by the death [71]. Blood pressure reaction of BHR to L-NAME administration can be seen even after the dose of 1 mg/kg/day within 3 weeks, while at similar dose normotensive rats remain unaffected [12, 36].

This short summary indicates that the response of normotensive and hypertensive organism to the same dose of L-NAME differs and, apparently, the sensitiv- ity of NOS/NO pathway is altered under hypertensive conditions [63, 70].

L-NAME – nitric oxide synthase activator?

Jana Kopincová et al.

Tab. 1. The effect of different dose of L-NAME and the treatment duration on the development of left ventricular hypertrophy in normotensive rats

Dose (mg/kg/day) Treatmentduration Strain Hypertrophy Reference

10 3 weeks Wistar A 77

10 3 weeks WKY P 19

20 25 days Wistar P 35

20 4 weeks Wistar P 58

40 4 weeks Wistar P 5, 10

50 8 weeks Wistar A (19 from 25) 3

50 8 weeks Wistar P (6 from 25) 3

~60 8 weeks Wistar P 87

Legend: A – absent, P – present, WKY – Wistar-Kyoto rats

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While L-NAME administration aimed at lowering of NO production is widely used, much less is known about the feedback consequences of long-term L-NAME treatment on NO production.

The phenomenon of feedback regulation of nitric oxide synthase expression and activity by the level of produced NO in vitro has already been observed in 1992 (for more details see [37]). Briefly, under physiological conditions, NO produced by NOS binds to heme iron of NOS molecule and thus inhibits syn- thetic activity of the enzyme. The rate of inhibition depends on isozyme of NOS. Accordingly, lowering NO levels in reaction solution by NO scavenging (e.g.

by oxyhemoglobin) led to maintaining of synthetic activity, while adding NO donor to the solution gradu- ally slowed down NO production [65]. In the same di- rection, expression of particular isoform of NOS is regulated by NO production. Preincubation of cell culture with exogenous NO donor led to attenuated expression of both inducible (iNOS) and endothelial NOS (eNOS) isoforms [16, 81]) and, on the contrary, the presence of NO scavenger increased iNOS expres- sion after cytokine stimulation [53].

The presence of L-NAME acts on NOS activity and expression in a similar manner like NO scavenging.

The presence of L-NAME in cytokine stimulated cell line led to both heightened expression and activity of iNOS [53] and, interestingly, incubation of the endo- thelial cells with L-NAME causes 140-percent en- hancement in eNOS protein level as an in vitro response to shear stress, which is a natural stimulus for NO synthesis in vivo [23]. However, it is rarely consid- ered that the same effect on NOS expression or activity may be reached in long term L-NAME treatment.

L-NAME and NOS activity

The first in vivo observation of L-arginine analogue- induced improvement of NO production was made by 7-day-lasting administration of 300 mg/kg/day of L-NMMA to Sprague-Dawley rats via continuous in- fusion through the portal vein by an osmotic pump [42]. As a result, increased iNOS expression in the liver tissue and 38-percent elevation in iNOS activity were achieved.

tal results. Not only in our laboratory, decreased NOS activity was repeatedly found after 4-week L-NAME administration of 40 mg/kg/day in the aorta, several brain parts, left ventricle or kidney [10, 11, 13, 58, 77], which was associated with worsen relaxant re- sponse of the aorta, renal and pulmonary artery to acetylcholine [28] – that is, after all, the original aim of the L-NAME usage.

By contrast, 7 weeks of L-NAME administration in the dose of 40 mg/kg/day to normotensive Wistar- Kyoto (WKY) rats led to increased NOS activity in the rat heart, aorta and kidney [59]. Amazingly, at the same dose of L-NAME, the treatment period and the rat strain, these authors found no change in NOS ac- tivity in another experiment [82], while decrease in NOS activity in the left ventricle and kidney of Wistar rats after 5 and 6 weeks of 40 mg/kg/day L-NAME treatment was found, together with impaired acetylcho- line-induced relaxation of the femoral and small mes- enteric arteries [55, 72]. Thus, it remains unanswer- able, why feedback regulation did not exhibit in one of experiments, and whether one week may serve as

“breaking point” in the molecular response to inhibitor or it is just a difference between two rat strains.

Aimed at verifying the existence of L-NAME-

induced feedback regulation of NOS activity in vivo,

we made several experiments on long-term L-NAME

treatment, using very low doses of L-NAME up to

0.3 mg/kg/day and prolonged treatment period (4 to

10 weeks). In the first experiment, normotensive Wis-

tar rats were treated by L-NAME in the dose of

1.5 mg/kg/day for 8 weeks and such treatment led to

increased NOS activity in the left ventricle and aorta

associated with improved response of the femoral ar-

tery to acetylcholine, while the blood pressure reac-

tion to L-NAME was only transient [9]. However, at

the same dose we did not find any elevation in NOS

activity in BHR with Wistar father [38]. Recom-

mended by reviewers, we switched to another nor-

motensive strain – WKY rats – and tried to lower the

dose with the aim to prevent the transient blood pres-

sure elevation. Ten weeks of L-NAME administration

of 0.3 and 1 mg/kg/day did not affect blood pressure,

but increased NOS activity in both the left ventricle

and aorta. Lowering of the dose and the strain change

showed up in NOS activity response to L-NAME

treatment also in BHR with WKY father. After 10-

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week treatment, the left ventricular and renal NOS ac- tivity were elevated within the dose of 0.3 mg/kg/day and aortal and renal NOS within 1 mg/kg/day of L-NAME (Fig. 1A, B, C [36]). However, comparing the effect of 4 and 10 weeks of 1 mg/kg/day of L-NAME treatment on NOS activity, we found that after 4 weeks there was reduction in NOS activity in the hypothalamus, aorta and liver [12] (see Tab. 2).

Thus it seems that feedback-regulated activation of NOS activity in vivo requires specific period of time.

When trying to evoke such feedback-induced NOS activity improvement in SHR, we were forced to ad- ditional dose cut-down, as SHR were not able to cope with 1 mg/kg/day of L-NAME. Thinking about the doses up to 100 mg/kg/day, it seemed irrational to ex- pect any results when treating SHR with the dose of 0.1 mg/kg/day. However, such low amount of L-NAME managed to elevate NOS activity in the left ventricle

and kidney of SHR (Fig. 1A, C [36]). So the results suggest that feedback control of NOS activity works both in normotensive and hypertensive rats. However, the sensitivity of such regulation showed between- strain and between-organ differences.

The only agreement in long-term L-NAME treat- ment effect consists in observation of decreased NOS activity in selected parts of the central nervous sys- tem. Administration of the dose from 0.1 or 1 mg/kg/

day to 40 mg/kg/day for 4 to 10 weeks caused rather inhibition than feedback-associated enhancement in brain NOS activity in both normotensive and hyper- tensive strains. This event was seen in spite of feed- back NOS activation exhibited in the peripheral tis- sues, suggesting that there is strong difference be- tween NOS activity regulation in the central nervous system and in the peripheral organs or tissues [32, 36, 59, 77] (Fig. 1D).

Fig. 1. The effect of ten week lasting low dose L-NAME treatment on NOS activity in the left ventricle (A), aorta (B), kidney (C) and hypothala- mus (D) of rats. White columns stand for controls, crosshatched columns stand for L-NAME dose 0.3 mg/kg/day, grey columns stand for L-NAME dose 0.1 mg/kg/day and black columns stand for L-NAME dose 1 mg/kg/day for 10 weeks. Total NOS activity was measured in tissue homogenates by determination of [³H]-L-citrulline formation from [³H]-L-arginine, as described previously [63] and expressed as pmol/min/mg of tissue proteins.⁺p < 0.05 compared to untreated controls; WKY – Wistar-Kyoto rats, BHR – borderline hypertensive rats, SHR – spontaneously hypertensive rats [36]

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L-NAME and NOS expression

Considering that L-NAME administration blocks NO synthesis, we can suppose that higher NOS activity found in particular tissues ex vivo results from higher level of NOS gene expression. Indeed, the presence of L-NAME increases in vitro the rate of both iNOS and eNOS expression [23, 53], perhaps via influencing the balance between NO and transcription factor nuclear factor kB (NF-kB) which is key factor in NOS ex- pression regulation [23, 30, 37]. NO is considered to inhibit either NF-kB activation [16], its binding to DNA [54] or to induce and stabilize NF-kB inhibitor [17, 61], and thus to modulate the rate of NOS tran- scription. In this sense, the decrease in NO level in- duced by L-NAME leads to NF-kB activation and consequently to increased NOS expression.

Paradoxically, it seems that the effect of L-NAME administration in vivo on NOS expression may differ depending on the treatment period and the tissue in- vestigated. Forty mg/kg/day of L-NAME for 4 weeks significantly increased eNOS and iNOS expression in Sprague-Dawley rat cavernous tissue [24] and eNOS in the Wistar rat heart and kidney, but it did not affect eNOS expression in the rat brain [59, 60]. However, the same dose of L-NAME did not affect eNOS or iNOS expression in Wistar aorta after 5 weeks of treatment [55], while 7 weeks of treatment potentiate eNOS expression increase in the rat heart and, con- trary, decreased eNOS expression in the rat brain [59].

Nevertheless, L-NAME-induced feedback regula- tion of NOS expression is more complicated than regulation of NOS activity. We have to reason out that activation of NF-kB may be evoked by several

the same way as NO deficiency and the presence of reactive oxygen species (ROS) may thus evoke higher eNOS or iNOS expression [22, 88]. Thereby, it is sometimes impossible to resolve, whether the increase in NOS expression is caused by NO depletion or rather by ROS-mediated NF-kB activation. Moreover, under conditions of oxidative stress, some of NOS co- factors turn inactive and NOS enzyme synthesizes rather superoxide radical than NO [14] which yet more worsen the vasoactive balance.

In addition, high-dose of L-NAME treatment (at least 40 mg/kg/day) is often associated with the ele- vated level of secondary products of oxidative stress [55, 82], and the same goes for spontaneous and bor- derline hypertension [33, 41, 75, 84]. This may be the background of higher eNOS expression, which was found repeatedly in BHR and in SHR even in the pre- hypertensive age of 3 weeks, suggesting that elevated blood pressure per se is not the cause of eNOS ex- pression increase [79, 80]. Hence, marked oxidative load after administration of high dose of L-arginine analogues may also be responsible for activation of iNOS expression and through that also iNOS activity in above mentioned experiment with 300 mg/kg/day of L-NMMA to Sprague-Dawley rats [42].

Secondly, NF-kB activation in the vascular tissue is either induced by shear stress [23], which is better known to activate eNOS via its phosphorylation [34].

But, however, shear stress may be accentuated due to blood pressure elevation after L-NAME administra- tion and thus it may activate NF-kB, which in turn may lead to increased eNOS transcription as well.

Nevertheless, increased NOS expression need not be associated with better NO bioavailability in vivo.

Under conditions of higher oxidative load, cofactors needed for NO synthesis, especially tetrahydrobio- pterin, may be oxidized and thus inactive [45] and NO may be scavenged by ROS [46, 75]. Despite marked NOS expression, in vivo activity of NOS could hence be diminished resulting in reduced NO bioavailability.

Thus, it is worthy to mention that this disparity be- tween NOS expression and activity entails pitfall in determination of NOS activity in vitro, as this method always requires adding of external cofactors to the re- action mixture and thus may reveal enzymatic acti- vity, which is absent in vivo. On that score, blood pressure in high-dose L-NAME-treated rats often re-

NOS activity L-NAME

4 weeks

L-NAME 10 weeks

Hypothalamus ¯ ¯

Aorta ¯

Liver ¯

Kidney 0

Legend: Direction of change is indicated:¯ – decrease in NOS activ- ity, – increase in NOS activity, 0 – no change in NOS activity [12]

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mains elevated despite observed NOS activation in vi-

tro

[59, 76] and likewise, vascular responses to vaso- dilatory agents often fail to exhibit any improvement [76].

In conclusion, this short review of literature on the effects of L-arginine analogues, especially L-NAME, showed that regulation of NO production in vivo is precisely regulated, but not precisely known process.

Disruption of this regulation by using apparent inhibi- tors of NO production and modulation of NO levels might paradoxically increase NOS expression or NOS activity depending on conditions of experiment – mainly on duration of treatment and dose of L- NAME, rat strain, tissue investigated as well as iso- form of NOS. This should be always taken into con- sideration when analyzing and discussing results re- garding the role of NO in physiological and patho- physiological conditions.

Acknowledgments:

This study was supported by Project “Center of Excellence for Perinatology Research” No. 26220120016 co-financed from EU sources, by Grant APVV-0523-10 and Grant VEGA No. 1/0291/12.

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Received: October 24, 2011; in the revised form: January 27, 2012; accepted: February 14, 2012.